Bovine serum albumin standard set kit

Cardiac mitochondria were isolated from guinea pig hearts using differential centrifugation as described in Wollenman et al.^{85 (link)} Briefly, Hartley albino guinea pigs weighing 350–450 g (4–6 weeks) were injected with heparin (500 units/mL) into the intraperitoneal cavity to prevent blood clotting during the cardiac mitochondrial isolation. Before heart removal, the animals were deeply anesthetized with 4–5% isoflurane. Prior to decapitation by guillotine, a noxious stimulus (paw pinch and eyelid reflex) confirmed the animals were fully sedated. After decapitation, a thoracotomy was performed. The heart was then perfused with cold cardioplegia solution and homogenized as described previously^{85 (link)}. Mitochondrial protein content was quantified using the BIO-RAD Bovine Serum Albumin (BSA) Standard Set Kit and the BCA assay. The mitochondrial suspension was diluted to a working concentration of 40 mg/mL and kept on ice for the duration of the experiment (4–8 h). Substrate stock solutions were neutralized to pH 7.0.

Cardiac mitochondria were isolated from guinea pig hearts using differential centrifugation, as described previously [8 (link),14 (link),22 (link)]. Briefly, Hartley albino guinea pigs weighing 350–450 g (4–6 weeks) were injected with heparin (500 units/mL) in the intraperitoneal cavity. The animals were anesthetized with 4–5% isoflurane and decapitated by a guillotine. After decapitation, a thoracotomy was performed, and the heart was perfused with a cold cardioplegia solution and homogenized using a handheld electronic homogenizer at 18,000 rpm for 20 s. The mitochondrial protein content was quantified using the BIO-RAD bovine serum albumin (BSA) standard set kit and the bicinchoninic acid (BCA) assay. The mitochondrial suspension was diluted to a working concentration of 40 mg/mL and kept on ice for the duration of the experiment (2–3 h). The substrate stock solutions were neutralized to a pH of 7.0.